Abstract: One of the recent paradigm shifts in stem cell biology and regenerative medicine has been the discovery that stem cells can begin to differentiate into adult tissue cells when exposed to intrinsic properties of the extracellular matrix (ECM), such as matrix structure, elasticity, and composition. ECM regulation of stem cells has also been shown to be as sensitive as well-studied soluble growth factors, and together in the body, they comprise the stem cell niche, or "microenvironment." However, these cues have typically been studied as isolated stimuli where no single cue, whether a growth factor or an ECM property, has been sufficient to generate the appropriate type of differentiated cells for a given regenerative cell therapy. Moreover, as stem cells mature in the body during development, their microenvironment is highly spatially- and temporally-controlled, yet our ability to dynamically regulate the niche as the body does has not be developed and is probably a critical requirement for developing differentiated cells from stem cells. Therefore, I propose to substantially advance the field of stem cell biology by developing a new hybrid hydrogel system using a unique combination of conventional polymer chemistries. These gels, comprised of hyaluronic acid-co-acrylamide polymer, should present spatially- and temporally-controlled matrix properties that mimic their presentation during development. When combined with spatially-patterned growth factors, these cues could more accurately recapitulate the development of a specific tissue ex vivo, which may improve the differentiated cell sources used for cell-based therapeutic applications. Public Health Relevance: Stem cells may hold the key to replacing cells and tissues lost in many devastating diseases, injuries, or by aging. Regulating how stem cells develop from immature, dividing cells to specific, non-dividing adult cells or tissues is critical to ensure proper development of therapeutic strategies to combat these medical problems. Much of our knowledge on how to direct stem cell maturation has focused on cell-secreted chemical signals, called growth factors. Recent studies have surprisingly shown that the extracellular matrix (ECM), a protein scaffold to which cells adhere, can also regulate stem cell development as well via a variety of properties that it can display, such as ECM elasticity, cell- ECM binding, cell shape, etc. Stimulation by either chemical or ECM-based cues using conventional materials, however, does not result in fully mature cells, and thus their suitability to be used to produce adult or "pre-programmed" stem cells for a clinically viable therapy is limited. Moreover, stem cells in the body are exposed to these cues to varying degrees based on their location within the organism as well as when the cues are expressed developmentally. Thus dynamic presentation of these cues also likely influences their ability to direct stem cells to become adult cells or tissues. Therefore, the investigations proposed here hypothesize that a combination of these cues, displayed at the developmentally appropriate time, place, and concentration using "smart" materials, would better recreate the microenvironment seen by cells in a given tissue. Improved mimicry could then induce more complete development of stem cells into adult cells as each signaling pathway within that cell that is required to make a specific cell or tissue type would start at the appropriate time. This form of "preprogramming" stem cells could improve their use in engineered tissues as cells could develop more naturally or in cell-based therapies as cells could possibly remodel the diseased microenvironment, such as the abnormally rigid scar formed after a heart attack.